Abstract
We previously demonstrated that the translocase protein TSPO2 together with the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT) were involved in a membrane transport complex in human red blood cells (RBCs). Because VDAC was proposed as a channel mediating ATP release in RBCs, we used TSPO ligands together with VDAC and ANT inhibitors to test this hypothesis. ATP release was activated by TSPO ligands, and blocked by inhibitors of VDAC and ANT, while it was insensitive to pannexin-1 blockers. TSPO ligand increased extracellular ATP (ATPe) concentration by 24–59% over the basal values, displaying an acute increase in [ATPe] to a maximal value, which remained constant thereafter. ATPe kinetics were compatible with VDAC mediating a fast but transient ATP efflux. ATP release was strongly inhibited by PKC and PKA inhibitors as well as by depleting intracellular cAMP or extracellular Ca2+, suggesting a mechanism involving protein kinases. TSPO ligands favoured VDAC polymerization yielding significantly higher densities of oligomeric bands than in unstimulated cells. Polymerization was partially inhibited by decreasing Ca2+ and cAMP contents. The present results show that TSPO ligands induce polymerization of VDAC, coupled to activation of ATP release by a supramolecular complex involving VDAC, TSPO2 and ANT.
Highlights
Human erythrocytes release ATP following exposure to β-adrenergic stimulation, mechanical deformation, reduced oxygen tension, or acidosis[1]
We investigated whether voltage dependent anion channel (VDAC) in close association with TSPO2 and adenine nucleotide transporter (ANT) acted as an ATP-release pathway at the red blood cells (RBCs) membrane and analysed upstream mechanisms involved in VDAC activation
We recently identified a macromolecular complex of approximately 800 kDa in the plasma membrane of these cells formed by VDAC, TSPO, ANT and other unidentified proteins, which was activated by TSPO ligands[23]
Summary
Human erythrocytes release ATP following exposure to β-adrenergic stimulation, mechanical deformation, reduced oxygen tension, or acidosis[1]. Leal-Denis et al.[12] showed that ATP release from RBCs induced by the peptide Mastoparan-7, a Gi-βγ protein activator, was only partially inhibited by the addition of CBX or PBC. Mathematical modelling of these results was compatible with ATP release by both pannexin-1 and a second conduit, whose nature remained elusive[9,12]. Studies using RBCs from pannexin-1-deficient mice showed a similar pattern, since ATP release from RBCs exposed to Mastoparan-712 or hypotonic K+ solutions[19] was only partially inhibited in pnx−/− knock out mice compared to pnx+/+ mice
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